Project Summary A variety of hematological diseases and malignancies are caused by an underlying dysfunction in hematopoietic stem and progenitor cells (HSPCs), the cells responsible for the maintenance of the blood system. Gold standard treatment for many of these malignancies is the effective replacement of the diseased cells via a hematopoietic stem cell transplant (HSCT), in which healthy donor hematopoietic stem cells (HSCs) self-renew, expand and give rise to hematopoietic progenitor cells (HPCs) that maintain all blood production for the lifetime of the individual. However, HSCTs are limited by availability of donor-matched cells and the low efficiency of ex vivo expansion of human HSPCs. Other hematological disorders and acute hematological trauma can be treated by transfusion of mature hematopoietic cells such as red blood cells and platelets; however, these treatments are also limited by donor supply. I hypothesize that SH2B3, an established negative regulator of cytokine signaling, HSC self-renewal and HSPC proliferation in mice, plays a similar role in humans and that inhibition of SH2B3 could be used to expand human HSPCs. This proposal will use two sources of HSPCs to comprehensively identify roles for SH2B3 in human hematopoiesis and test depletion of SH2B3 as a strategy to expand human HSPCs. In Aim 1 I will use HPCs derived from human pluripotent stem cells (hPSCs). The cytokine directed differentiation of hPSCs to HPCs closely follows stages of hematopoietic development and the HPCs have erythroid, myeloid and megakaryocycitic lineage potential. Therefore, I will use SH2B3-/- hPSCs to help elucidate the role of SH2B3 in human hematopoietic development, in erythroid, myeloid and megakaryocytic lineage cells and to determine if targeting SH2B3 expression can augment the production of hematopoietic cells from hPSCs. Increasing hematopoietic cell production from hPSCs could be used for the in vitro manufacturing of mature hematopoietic cells for therapeutic transfusions. Differentiation of hPSCs to HPCs does not fully recapitulate human hematopoietic development and engraftable HSCs cannot be obtained from these cells yet. Therefore, in Aim 2 I will use primary human HSPCs isolated from umbilical cord blood (UCB). By disrupting SH2B3 expression in these cells I will be able to further identify cytokine signaling cascades in which SH2B3 functions in human hematopoiesis, and determine if SH2B3 depletion results in the ex vivo and in vivo expansion of functional HSCs, which will be useful for HSCTs. Together, these proposed studies will comprehensively identify roles of SH2B3 in human hematopoiesis and will test if SH2B3 deficiency causes an expansion of HSPCs to increase the supply of functional HSCs for use in HSCTs and mature hematopoietic cells for use in therapeutic transfusions. These studies will provide proof of concept for potential use of reversible inhibition of SH2B3 in the clinic.